A multiparametric calcium signal screening platform using iPSC-derived cortical neural spheroids.

Abstract

Discovery of therapeutics for neurological diseases is hampered by the lack of predictive in vitro and in vivo models. Traditionally, in vitro assays rely on engineered cell lines grown two-dimensionally (2D) outside a physiological tissue context, which makes them very amenable for large scale drug screening but reduces their relevance to in vivo neurophysiology. In recent years, three-dimensional (3D) neural cell culture models derived from human induced pluripotent stem cells (iPSCs) have been developed as an in vitro assay platform to investigate brain development, neurological diseases, and for drug screening. iPSC-derived neural spheroids or organoids can be developed to include complex neuronal and glial cell populations and display spontaneous, synchronous activity, which is a hallmark of in vivo neural communication. In this report we present a proof-of-concept study evaluating 3D iPSC-derived cortical neural spheroids as a physiologically-and pharmacologically-relevant high-throughput screening (HTS) platform and investigate their potential for use for therapeutic development. To this end, a library of 687 neuroactive compounds were tested in a phenotypic screening paradigm which measured calcium activity as a functional biomarker for neural modulation through fluctuations in calcium fluorescence. Pharmacological responses of cortical neural spheroids were analyzed using a multi-parametric approach, whereby seven peak characteristics from the calcium activity in each well were quantified and incorporated into principal component analysis and Sammon mapping to measure compound response. Here, we describe the implementation of the 687-compound library screen and data analysis demonstrating that iPSC-derived cortical spheroids are a robust and information-rich assay platform for HTS.

High-Throughput Screening Assay for Detecting Drug-Induced Changes in Synchronized Neuronal Oscillations and Potential Seizure Risk Based on Ca2+ Fluorescence Measurements in Human Induced Pluripotent Stem Cell (hiPSC)-Derived Neuronal 2D and 3D Cultures

“…However, with multi-parametric analysis, the outcome and conclusions can be complex and potentially confusing. For example, the significant effects of a positive control, 4-AP, were found to be within the data cluster of DMSO controls in 3D-hiPSC-derived neuron cultures in a study reported by others [ 37 ]. Further studies with additional reference drugs encompassing additional pharmacological profiles will need to be evaluated using this HTS Ca 2+ -transient assay within hiPSC-derived neurons to confirm if a single parameter (i.e., the peak number) is sufficient and optimal for the determination of drug-induced changes in neural oscillations and potential risk for seizure.…”

A human stem cell-derived neuronal model of morphine exposure reflects brain dysregulation in opioid use disorder: Transcriptomic and epigenetic characterization of postmortem-derived iPSC neurons

“…A study investigating the effect of methadone on iPSC-derived cortical organoids found disruption of neural growth and suppression of neural electrochemical activity ( 34 ). iPSC derived neural spheroids have been used to screen for neuromodulatory activity and identify drug targets for opioid use disorder ( 35 , 36 ). Other studies have investigated opioid roles in non-neuronal cell types including endothelial cells ( 37 , 38 ) and cardiomyocytes ( 39 ).…”

High content screening miniaturization and single cell imaging of mature human feeder layer-free iPSC-derived neurons

“…iPSC-derived neuron model systems are particularly attractive as physiologically relevant neurological models, although there has been a surprising lack of standardized maturation and miniaturization procedures for the in vitro systems especially when using low oxygen culturing conditions. However, iPSC-derived neurons have been used in miniaturized formats (i.e., 384 well assay plates) but they focus on using commercially available pre-plated, pre-formed spheroids [ 37 ] matured 9.5 weeks, bulk neuronal differentiation [ 38 ] or co-culturing with astrocytes for extended culturing [ 39 ]. Moreover, as mentioned previously, NeuN has not been commonly used as a neuronal maturation marker in the assay miniaturization process.…”